The sinoatrial node (SAN) is the primary pacemaker of the mammalian heart. With age, dysfunction of the SAN increases exponentially. We have documented a significant decrease in the intrinsic beating rate (IBR) of young (3-month) and old (24-month) C57Bl mice in last year. Last year, we found that the early onset of age associated reduction in the mouse SAN IBR is by 12 months, with no further reduction at older age to 24 months. We hypothesized changes in Ca2+ handling and PKA-dependent signaling might play a significant role in the age-related SAN dysfunction. Our lab has previously shown PDE inhibition not only elevates cAMP concentration in pacemaker cells but also leads to enhanced cAMP mediated-PKA-dependent phosphorylation of Ca2+ cycling proteins as indexed by phospholamban phosphorylation at serene 16 site. Our data shows the inhibition of the degradation of cAMP, by inhibition of degradation by phosphodiesterase using IBMX (Phosphodiesterase inhibitor) can raise the old SANs IBR to comparable levels that occur in the young SANs at high dose of IBMX (1 milli M). However, the IBR of SANs from old mice is less sensitive to IBMX mediated acceleration. The IC50 of IBMX for change in IBR is 2.8 micro M in young SANs (2 month) and 30 micro M in old SANs (20 month) (n=5 in each group, P<0.01). Concomitantly, the preliminary data on single cells dissociated from old SANs shows decreased phospholamban phosphorylation at serine 16 site (PKA site) at basal state and under IBMX stimulation, which is consistent with the decreased sensitivity to IBMX stimulated increase in IBR. This year we found that sinoatrial nodes from Aged C57 mice exhibit lower intrinsic beating rates. Sinoatrial nodal cells from Aged C57 mice have reduced SR Ca2+ loads and smaller LCRs and these are reduced in size, duration, and number. SANs and SANC isolated from aged C57 mice vs. young mice exhibit A reduced sensitivity to respond to an increase in cAMP-PKA-Ca2+ signaling due to PDE inhibition. These results suggest that the intrinsic cAMP-PKA-Ca2+ signaling is deficient in the aged mice. This deficiency may result from: 1.)reduced amount or function of Ca2+ cycling proteins e.g. SR Ca2+ pump, phospholamban (PLB), RyRs. and/or 2.) reduced phosphorylation of Ca2+ cycling proteins e.g.PLB, in response to an increase in cAMP-mediated-PKA dependent phosphorylation. 3.) These deficiencies may explain why the aged heart cannot beat as fast as the young heart. Future plans are: 1) to employ western blotting in cells from young and old SANs to measure Ca2+ cycling protein density; 2) to directly measure the downstream signaling of cAMP-PKA- Ca2+ pathway i.e. PKA-dependent protein phosphorylation and phosphatase activity.